Research Article Received: 19 September 2016 Revised: 6 January 2017 Accepted: 23 February 2017 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2017, 31, 791–803 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7846 Rapid quantification and isotopic analysis of dissolved sulfur species Derek A. Smith1*,† , Alex L. Sessions1, Katherine S. Dawson1, Nathan Dalleska2 and Victoria J. Orphan1 1Division of Geological and Planetary Sciences, California Institute of Technology, 1200 E California Blvd, Pasadena, CA 91125, USA 2Environmental Science and Engineering, California Institute of Technology, 1200 E California Blvd, Pasadena, CA 91125, USA RATIONALE: Dissolved sulfur species are of significant interest, both as important substrates for microbial activities and as key intermediaries in biogeochemical cycles. Species of intermediate oxidation state such as sulfite, thiosulfate, and thiols are of particular interest but are notoriously difficult to analyze, because of low concentrations and rapid oxidation during storage and analysis. METHODS: Dissolved sulfur species are reacted with monobromobimane which yields a fluorescent bimane derivative that is stable to oxidation. Separation by Ultra-Performance Liquid Chromatography (UPLC) on a C18 column yields baseline resolution of analytes in under 5 min. Fluorescence detection (380 nm excitation, 480 nm emission) provides highly selective and sensitive quantitation, and Time-of-Flight Mass Spectrometry (TOF-MS) is used to quantify isotopic abundance, providing the ability to detect stable isotope tracers (either 33Sor34S). RESULTS: Sulfite, thiosulfate, methanethiol, and bisulfide were quantified with on-column detection limits of picomoles (μM concentrations). Other sulfur species with unshared electrons are also amenable to analysis. TOF-MS detection of 34S enrichment was accurate and precise to within 0.6% (relative) when sample and standard had similar isotope ratios, and was able to detect enrichments as small as 0.01 atom%. Accuracy was validated by comparison to isotope-ratio mass spectrometry. Four example applications are provided to demonstrate the utility of this method. CONCLUSIONS: Derivatization of aqueous sulfur species with bromobimane is easily accomplished in the field, and protects analytes from oxidation during storage. UPLC separation with fluorescence detection provides low-μM detection limits. Using high-resolution TOF-MS, accurate detection of as little as 0.01% 34S label incorporation into multiple species is feasible. This provides a useful new analytical window into microbial sulfur cycling. Copyright © 2017 John Wiley & Sons, Ltd. Sulfur exists in the natural environment in myriad forms, oxygen minimum zones of the world’s oceans,[5,6] the low 2– fi ’ ’ [7,8] ranging in oxidation state from sulfate (SO4 ) to sul de sulfate methanogenic zone of marine sediments, salt – (HS ) and including many species of intermediate oxidation marshes,[9] freshwater sediments,[10,11] and within symbiotic fi – 0 [9,12] state, such as polysul de (HSx ), elemental sulfur (S ), microbial associations. In all cases, microbial sulfur thiosulfate, trithionate, sulfite, and others. Species such as cycling is thought to play key roles in the remineralization of thiosulfate, trithionate, and sulfite in particular are thought organic matter. to be important intermediaries in microbial reactions that Sulfur speciation also dramatically affects the solubility and – transform (either oxidize, reduce, or disproportionate) sulfur bioavailability of trace metals,[1 3] abiotic oxidation and and are frequently coupled to the biogeochemical cycles of reduction of manganese and iron,[1,4] and sulfurization of – carbon, oxygen, and iron.[1 4] ’Cryptic’ sulfur cycles driven organic matter.[13,14] Studies of all these processes, and others, by microbial metabolism have been hypothesized to occur in would benefit greatly from improved methods for quantifying dissolved sulfur species, particularly the most transient, intermediate compounds. Moreover, an ability to distinguish * Correspondence to: D. A. Smith, Department of Earth and stable isotope labels in such compounds would enable a Planetary Sciences, Washington University in Saint Louis, wide variety of stable isotope probing experiments that Campus Box 1169, Rudolph Hall Rm 110, 1 Brookings could help illuminate the cryptic microbial sulfur cycle. A Drive, St. Louis, MO 63130, USA. variety of techniques to measure dissolved sulfur species E-mail: [email protected] have previously been described. One of the most widely † Current address: Department of Earth and Planetary utilized techniques, the Cline assay, employs absorption Sciences, Washington University in Saint Louis, Campus spectrophotometry of a N,N-dimethyl-p-phenylene diamine, Box 1169, Rudolph Hall Rm 110, 1 Brookings Drive, St. chloride, and sulfide complex (methylene blue) to quantify 791 Louis, MO 63130, USA. hydrogen sulfide.[15] Rapid Commun. Mass Spectrom. 2017, 31, 791–803 Copyright © 2017 John Wiley & Sons, Ltd. D. A. Smith et al. Ion chromatography (IC) has been employed to separate have been previously used to quantify sulfur species from multiple anions of sulfur (i.e., [16,17]). However, IC eluent a range of diverse samples including hydrothermal vent fl 2– 2– chemistry can alter sulfur speciation; some sulfur species, uids, sulfur-oxidizing bacterial cultures (SO3 ,S2O3 ), – – e.g. polysulfides, are too reactive for accurate quantification HS in human blood, and cysteine and HS in – with IC; further, IC provides no isotopic information, methanogens.[27 31] although it has been combined with multi-collector To improve on the separation of derivatized sulfur species inductively coupled plasma mass spectrometry (MC-ICP- by HPLC, which takes ~60 min per sample,[24,25] we employ MS) to achieve this.[18,19] Derivatization of sulfur species with Ultra-Performance Liquid Chromatography (UPLC) for rapid methyl trifluoromethanesulfonic acid (methyl triflate) is (<5 min) separation of derivatized sulfur species. For capable of stabilizing polysulfides with quantification detection and quantitation, we evaluated both a conventional down to approximately 25 μM by UV absorption fluorescence detector and a state-of-the-art Time-of-Flight spectrophotometry coupled to high-performance liquid (TOF) mass spectrometer (TOF-MS). Fluorescence detection chromatography (HPLC); however, with extraction and is less expensive and more sensitive than MS, and evaporation post-extraction, lower limits are reported.[20] nondestructive so it can be coupled with preparative fraction Drawbacks of the methyl triflate method include the toxicity collection of individual sulfur species, e.g. for subsequent of the reagent, multistep extraction procedure, addition of high-precision analysis of natural abundance isotope ratios. sulfur to the derivatized product, and applicability only to However, fluorescence itself provides no isotopic information, reduced sulfur species. Cyclic voltammetry and in situ probes which is the main impetus for using MS. The high-resolution have also been used to measure and quantify many of the TOF-MS employed here allows us to distinguish isobaric reactive sulfur species in environmental samples at relatively interferences in the molecular ions, and accurately quantify high spatial resolutions (mms).[9,21,22] The limitations of cyclic 34S enrichment down to levels of ~0.6%. The same voltammetry are that it cannot distinguish between the methodological approach could presumably be used for 33S isotopes of sulfur. enrichments. We note that while TOF-MS was used in this Here, we adapt a previously described method for study, quadrupole or other low-resolution MS could be an preserving aqueous nucleophilic sulfur species as bimane alternative, with the caveat that isotopic discrimination might derivatives, and update their separation and detection to suffer. modern instrumentation. Key features of the derivatization To refine and test this updated methodology, we focused on 2– 2– – are that (1) reaction with bromobimane (mBBr) is four sulfur analytes: SO3 ,S2O3 ,CH3SH, and HS . Other quantitative thus causing no isotopic fractionation; (2) the sulfur species are also potentially accessible with this resulting bimane derivatives are very stable towards approach. These analytes were measured in standard subsequent oxidation, providing a convenient means to solutions (both a dilution series of decreasing concentration, preserve samples in the field; and (3) bimane is strongly and a series of varying isotopic composition), then fluorescent, enabling non-destructive fluorescence characterized in four separate proof-of-principle applications: – detection.[23 26] The quantitative mBBr reaction proceeds as (1) measurement of sulfite and thiosulfate concentrations in a nucleophilic substitution of sulfur compounds containing marine sediment pore-waters, where they are present at low an unshared pair of electrons (Fig. 1), which includes sulfite μM concentrations; (2) measuring 34S appearance in bisulfide 2– 2– fi (SO3 ), thiosulfate (S2O3 ), organic thiols (R-SH), bisul de in a pure culture of sulfate-reducing bacteria amended with – fi 2– 0 34 2– 34 fi (HS ), and polysul des (Sn ) but not elemental sulfur (S ) SO4 tracer; (3) measuring S in bisul de in methane seep 2– 34 2– or sulfate (SO4 ). mBBr is a neutral molecule capable of sediments incubated with SO4 tracer; and (4) identifying crossing the cellular membrane, enabling reactions with both previously unreported intermediate sulfur species in